2012 Annual Report
1a.Objectives (from AD-416):
1. The long-term goal of this Objective is to develop soybean seeds that have increased oil levels derived at the expense of non-structural carbohydrates. 2. Molecular biology techniques will be used to improve the nutritional quality of soybean seed proteins. 3. To develop the molecular basis for modification of the fatty acid components of soybean oil and anti-nutritional components in soybean meal to use in accelerated breeding programs. 4. Identify effects on key performance determinants of crop seed quality resulting from modified seed composition using traditional or non-traditional genetic methods.
1b.Approach (from AD-416):
To reach the overridging objective of the modification of soybean seed composition for food, feed, and industrial uses requires a team approach that spans the complete range from basic biochemical assessment of possible target sites to the evaluation of the agronomic properties and value of modified soybeans. Basic biochemical approaches will be used to assess the effect of manipulating the expression of a key enzyme complex that is at the interface of carbon partitioning into oil or carbohydrates. A proteomic approach to the analysis of soybean seed development will allow for the discovery of other key regulatory events that offer possibilities for manipulation. Transgenic approaches will be used to modify the protein content and constitution of the soybean seed such that the nutritional quality as feed can be improved. A similar approach combined with classical molecular genetic approaches to plant breeding will be directed at altering the fatty acid components and anti-nutritional compounds of soybean seed to improve not only the nutritive value of the seed but also the health aspects of soybean consumption. A classical physiological approach serves to address the efficacy of the targeted modifications as they relate to agronomic concerns of yield, seed quality, and storage.
The systems biology platform being assembled for analysis of soybean seed development has been largely based upon results from carbon-centric metabolism. This was initially reasonable because two of the main targets for modification are triacylglycerol and the raffinosaccharide anti-nutrients. Since protein quality is also a target it will be important to include at least some emphasis on nitrogen and sulfur metabolism. We have begun to address the former using the regulatory enzyme glutamine synthase as a probe. Preliminary results suggest that nitrogen/protein emphasis characterizes an earlier time-index position than carbon metabolism in seed development.
We overexpressed a key sulfur assimilatory enzyme, the cytoplasmic form of O-acetylserine sulfhydrylase (OASS) in soybeans. The transgenic plants have a 4- to 10-fold increase in OASS activity, and a 74% increase in protein-Cys levels (2.8 % total amino acid content) compared to non-transformed soybean seeds (1.6 % total amino acid content). A 32% increase in free Cys was also observed. Transgenic plants also had a marked increase in the Bowman-Birk protease inhibitor, a Cys-rich protein. The overall increase in soybean total Cys content will satisfy the nutritional requirements of monogastric animals.
A model was developed to explain the basis of genetic drag when introgressing seed composition traits into high yielding soybean cultivars. Distinguishing genetic drag from direct negative effects due to the consequences of the targeted alleles is important in understanding the potential success of a seed composition trait. Through three rounds of backcrossing, 20 lines were developed that captured the high oleic acid seed oil trait in a commercial soybean cultivar genomic background. Leaf tissue was collected from each of the lines for the isolation of DNA that will be characterized for genome contributions from the two parents.
Increasing stearic acid content of soybean seed oil is an alternative to partial hydrogenation. Stearic acid is normally present at trace amounts in seeds of conventional soybean lines (~3-5%). One gene deletion in soybean line ‘A6’ had previously been reported as solely causative for the elevation of seed stearic acid to 25.7±4.4%. A method to detect and quantify genomic deletions was used with four moderately elevated seed stearic acid (11-13%) radiation induced mutants. In contrast to expectation, we found that the same gene was deleted in all lines examined, but they differed in their stearic acid content as compared to ‘A6’. We conclude that a second genetic locus must be present in ‘A6’ which acts synergistically with the gene deletion.
Where soybeans are grown alters their human health potential. Growing location has a pronounced effect on the accumulation of the cancer chemopreventive agent Bowman-Birk inhibitor (BBI) in soybean seeds. BBI is a serine protease inhibitor which has been touted as a potential cancer chemopreventive agent for humans. ARS scientists in Columbia, Missouri examined the protein profile of eight soybean varieties grown in three Missouri locations and demonstrated that soybean varieties that were grown in one particular location contained reduced amounts of BBI in their seeds. These results demonstrate that growing location can modulate the concentration of this cancer chemopreventive agent and thus its level can be improved by simple changes in agronomic practices.
New soybean low in the anti-nutritional compound phytic acid released. Conventional soybean seeds possess high levels of the essential nutrient phosphorus. However, the majority of this phosphorus is present in the form of phytate, which cannot be effectively digested by pigs, horses, and humans. The undigested phytate can lead to environmental pollution problems in water run-off from livestock operations using soybeans as feed. The causative genetic mutations in two soybean mutant lines with decreased levels of phytate were recently discovered. ARS scientists in Columbia, Missouri used genetic tracking tools in a genetic crossing scheme to identify new lines that had unprecedented reductions in phytate. The new lines had no overall change in total phosphorus content, suggesting that they have higher phosphate bioavailability than any currently available lines and can be used to develop commercially useful soybean varieties with a broader applicability for feed and lower environmental impact.
Natural chemical modification of proteins. Natural post-translational modification (PTM) of proteins is a large and very important aspect of proteomic analyses. Historically, protein phosphorylation (addition of a phosphate group) and N-glycosylation (addition of a sugar molecule) are the PTM that have received the most attention. In recent years, however, it has become increasingly obvious that acetylation of the amino acid lysine in a protein (abbreviated to PKA) is a widespread PTM and can drive regulation of gene expression and metabolic activity. ARS scientists in Columbia, Missouri developed a new strategy for detection of PKA in developing soybean seeds. Employing this strategy unequivocally identified hundreds of acetylated soybean proteins, which can be sorted into regulatory and metabolic categories. This information provides a starting point for analysis of the mechanisms by which protein PTM control seed development and composition, ultimately leading to improved crops for the benefit of producers and consumers.
Casteel, J., Miernyk, J.A., Thelen, J.J. 2011. Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis. Plant Physiology and Biochemistry. 49:1355-1361.
Kim, W., Jang, S., Krishnan, H.B. 2011. Accumulation of leginsulin, a hormone-like bioactive peptide, is drastically higher in Asian than in North American soybean accessions. Crop Science. 52:262-271.
Gillman, J.D., Tetlow, A., Deong-Lee, J., Shannon, G.J., Bilyeu, K.D. 2011. Loss-of-function mutations affecting a specific Glycine max R2R3 MYB transcription factor result in brown hilum and brown seed coats. Biomed Central (BMC) Plant Biology. 11:155. Available: http://www.biomedcentral.com/1471-2229/11/155.
Palavalli, M.H., Natarajan, S.S., Wang, T.T., Krishnan, H.B. 2012. Inhibition of soybean seeds in warm water results in the release of copious amounts of Bowman-Birk protease inhibitor, a putative anticarcinogenic agent. Journal of Agricultural and Food Chemistry. 60:3135-3143.
Wang, T.T., Boue, S.M., Krishnan, H.B. 2011. The protective effect of soybean phytochemicals on androgen responsive human prostate cancer cells LNCaP is likely mediated through modulation of hormone/cytokine-dependent pathways. Functional Foods in Health and Disease. 1(11):457-471.
Lee, J., Bilyeu, K.D., Pantalone, V., Gillen, A.M., So, Y., Shannon, J. 2012. Environmental stability of oleic acid concentration in seed oil for soybean lines with FAD2-1A and FAD2-1B mutant genes. Crop Science. 52(3):1290-1297.
Anh-Tung, P., Jeong-Dong, L., Grover, S.J., Bilyeu, K.D. 2011. A novel FAD2-1 A allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content. Journal of Theoretical and Applied Genetics. 123(5):793-802.
Boersma, J.G., Ablett, G.R., Grainger, C., Gillman, J.D., Bilyeu, K.D., Rajcan, I. 2012. New mutation in Delta-9-Stearoyl-Acyl Carrier Protein desaturase gene associated with enhanced stearic acid levels in soybean seed. Crop Science. 52:1736-1742.
Krishnan, H.B., Jang, S., Baxter, I.R., Wiebold, W.J. 2012. Growing location has a pronounced effect on the accumulation of cancer chemopreventive agent Bowman-Birk inhibitor in soybean seeds. Crop Science. 52:1786-1794.
Miernyk, J.A. 2012. “Parts Is Parts,” at least from the proteomics perspective. In: Mock, H-P., Wang, Zz-Y, editors. Frontiers in Agriculture Proteome Research.Tsukuba, Japan: Tsukuba Science City Press. p. 24-28.
Ashan, N., Swatek, K.N., Zhang, J., Miernyk, J.A., Xxu, D., Thelen, J.J. 2012. “Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex. Frontiers in Plant Proteomics. 3:1-10.